Classical methods for determining the presence and number of microorganisms in a sample are time consuming, tedious and labor intensive. Typically, a technician must prepare reagents and nutrients, mix the nutrients with agar, heat the mixture, pour the mixture into a petri dish, allow the agar to gel, obtain a test sample, dilute the test sample, add an aliquot of the diluted sample to the agar, incubate the inoculated plate for 24-48 hours and finally count the number of microbial colonies growing in the petri dish. Products and processes which reduce the preparation time and which allow an earlier, more rapid detection and count of these microorganisms, such as bacteria, would clearly be welcomed by those working in this field.
One example of a product which greatly simplifies the above preparation time is a thin film, dry culture device for growing microorganisms that is described in U.S. Pat. No. 4,565,783 to Hansen et al. In a typical thin film device reported by Hansen et al., a cold-water soluble dry powder containing a gelling agent and microbial growth nutrients is coated on a waterproof substrate. A transparent, read-through cover sheet coated on a surface with an acrylate adhesive containing an indicating dye and powdered gelling agent is attached to the coated substrate. A thin film, dry culture plate device based on the report of Hansen et al. for enumerating coliform bacteria in a sample is commercially available as PETRIFILM plates (Catalog No. 6400, 3M, St. Paul, Minn.
When the thin film, dry culture plate device is used, a predetermined amount of an aqueous sample is typically placed in contact with the coated substrate and the cover sheet is placed over the sample and substrate. The aqueous sample hydrates the soluble dry powder which then forms a gelled medium capable of sustaining microbial growth. During the growth period, the indicator dye adhered to the cover sheet reacts in the presence of viable microorganisms to give a detectable response that allows visualization of microbial colonies growing on the culture device.
The dry thin film, dry culture plate devices of Hansen et al. are much simpler to use than conventional gelled agar medium/petri dish systems because there is no need for the user to heat and mix the growth medium, agar and other reagents and then add the mixture to petri dishes or pour plates. In addition, the devices of Hansen et al. are compact and easily disposed of and therefore are easier and safer to use. A number of variations and/or modifications of the thin film plates of Hansen et al. have also been reported. For example, Hill et al., U.K. patent application 2 177 419 A, report use of a specific diluent solution to selectively grow lactic acid bacteria on PETRIFILM plates, Nelson et al., U.S. Pat. No. 5,089,413, report a modified thin film device adapted to grow aerobic microorganisms, Suzuki et al., U.S. Pat. No. 5,147,801 report a modified thin film device containing a wet hydrophilic layer or sheet on a water repellant substrate, and Nelson et al., U.S. Pat. No. 5,232,838, report a thin film device incorporating a water-based adhesive to allow use of greater amounts of a sample on the device.
In spite of the many advantages that the thin film, dry culture plates of Hansen et al. and related devices have over conventional types of culture systems, the inoculated thin film plates must still be incubated for 24-48 hours before the number of microbes may be determined. The ability to detect the presence or determine the number of microbes, particular bacteria, at an earlier time may be highly desirable and very valuable in many circumstances.
For example, earlier detection and rapid enumeration of bacteria in a selected sample is important in the food industry. At the present time, the enumeration of bacteria in a sample after an incubation time of 24-48 hours requires processors to delay distribution of food products and may allow the production of large amounts of contaminated food products. Earlier detection of bacteria in food products would allow the processor to release food products for distribution more quickly because contamination or lack of contamination could be established at an earlier time. In addition, a processor might be able locate and correct a source of bacterial contamination without having to discard large amounts of contaminated food products. Thus, detection and enumeration of bacterial contamination in less than 24-48 hours would be extremely beneficial to food product producers. See, e.g., Phebus et al., Journal of Rapid Methods and Automation in Microbiology, 1:249-260 (1993) that report use of OXYRASE membrane fraction to shorten the lag phase period of growth of pathogenic Escherichia Coli 0157:H7 (a pathogen associated with a 1993 food poisoning outbreak caused by eating undercooked ground beef).
Although the food industry would clearly benefit by determining microbial contamination at an earlier time, other industries would also welcome the opportunity to detect and/or enumerate microorganisms quickly. A need exists for products and processes which allow the early detection and rapid count of microorganisms.